JP2009109402A - Image information processing apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an image information processing apparatus for differentiating between the fluorescent luminance of a probe and the luminance due to a foreign substances adhering to the probe array surface, and defective pixels existing in an area sensor, when the fluorescent brightness of a probe on a probe array, such as DNA micro array, is detected. <P>SOLUTION: This image information processing apparatus detects the fluorescent luminance of the probe on the probe array, based on a hybridization reaction result, while differentiating it from the fluorescent luminance of other than the probe in a hybridization-reacted image of the probe array. The image information processing apparatus comprises a means for calculating the luminance ratios of red, green and blue pixels in the hybridized image, and a means for differentiating between the fluorescent luminance of the probe and the luminance of other than the probe, based on the luminance ratios of the red, green and blue pixels. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a so-called probe array image information processing apparatus.

2. Description of the Related Art Conventionally, there are sensors using a CCD sensor, a CMOS sensor, or the like as an area sensor used in an image pickup apparatus. These amplify signal charges accumulated in a photodiode, which is a photoelectric conversion element of each pixel, by each method, and read them out as image information. Patent Document 1 discloses a nucleic acid analyzer that reads the fluorescence of a DNA microarray using a CCD sensor. In the apparatus disclosed in this patent document, each probe of the DNA microarray is excited by irradiation with a light emitting diode, and the fluorescence of each probe is detected by a CCD sensor. This apparatus makes it possible to measure the fluorescence intensity of each probe of the DNA microarray at a time. Furthermore, the measurement conditions of each probe are almost the same, and the measurement accuracy is improved. Further, the reading mechanism is space-saving and inexpensive, the failure rate is reduced, and maintenance is almost unnecessary.
JP 2005-181145 A

  On the surface of the DNA microarray substrate, foreign matter such as dust and dirt may adhere in addition to the probe. In the area sensor, there are defective pixels that cannot read normal signal charge information, such as when a defect occurs in the photodiode of the pixel. For this reason, when the fluorescence image obtained by the probe hybridization reaction of the DNA microarray is captured by the area sensor, the brightness of the fluorescence image is detected not only by the fluorescence brightness of the probe but also by the foreign matter or the abnormal value of the defective pixel as the brightness. Is done. Therefore, in order to detect the fluorescence luminance of the probe, it is desirable to distinguish only the fluorescence intensity of the probe by distinguishing the fluorescence luminance of the probe from the luminance due to the foreign matter or defective pixel.

  The object of the present invention is to distinguish between the fluorescence intensity of a probe and the brightness caused by a foreign pixel adhering to the probe array surface or a defective pixel existing in an area sensor when detecting the fluorescence intensity of a probe on a probe array such as a DNA microarray An object of the present invention is to provide an image information processing apparatus that can perform such processing.

  Another object of the present invention is to provide an image information processing apparatus that interpolates pixels with saturated luminance in a hybridized image of a probe array.

  It is a further object of the present invention to provide an image pickup apparatus for a probe array comprising the image information processing apparatus.

The first image information processing apparatus of the present invention is
An image information processing apparatus for detecting the fluorescence intensity of probes on a probe array based on the hybridization reaction result separately from the fluorescence intensity other than the probe in an image obtained by performing a hybridization reaction of a probe array,
Means for calculating a luminance ratio of red, green and blue pixels in the image;
Means for distinguishing the fluorescence brightness of the probe and the fluorescence brightness other than the probe from the brightness ratio;
An image information processing apparatus comprising:

The first image capturing apparatus of the present invention is
An apparatus for taking an image of a probe array hybridization reaction,
Probe array holding means;
Means for irradiating the probe array with excitation light;
An area sensor for detecting fluorescence intensity on the probe array;
A first image information processing apparatus of the present invention;
It is a probe array image pick-up device provided with.

The second image information processing apparatus of the present invention is
An image information processing apparatus that interpolates pixels with saturated brightness in a hybridized image of a probe array,
Means for calculating a luminance ratio of red, green and blue pixels in the image;
Means for calculating the luminance of the pixel whose luminance is saturated from the luminance ratio;
An image information processing apparatus comprising:

The second image capturing apparatus of the present invention is
An apparatus for taking an image of a probe array hybridization reaction,
Probe array holding means;
Means for irradiating the probe array with excitation light;
An area sensor for detecting fluorescence intensity on the probe array;
A second image information processing apparatus of the present invention;
It is a probe array image pick-up device provided with.

  According to the present invention, in the probe array hybridization image, the fluorescence intensity of the probe is distinguished from the brightness due to foreign matters such as dust and dirt adhering to the probe array, and the abnormal brightness due to the area sensor defective pixel. It becomes possible to detect the reaction result of a simple probe, for example, the fluorescence luminance.

  FIG. 1 is a flowchart showing a procedure of an image information processing method according to the present invention. First, a fluorescence image obtained by hybridization reaction of a DNA microarray is picked up by an area sensor (101). Since the fluorescent image is picked up using an area sensor equipped with an RGB color filter, it is possible to obtain image information composed of signals of RGB wavelengths. Next, object recognition is performed from the brightness level of the image information (102). An object is an area having a higher luminance than the background. The object includes probe fluorescence brightness of the DNA microarray, brightness due to foreign matters such as dust and dirt adhering to the DNA microarray substrate, abnormal brightness due to defective area sensor pixels, and the like. Here, the probe of the DNA microarray includes a control probe that emits fluorescence intensity for any sample. Next, the RGB luminance ratio of the object is calculated (103). The ratio of each luminance of R, G, and B included in the object area is calculated and obtained. Next, the RGB luminance ratio of the control probe object is compared with the RGB luminance ratio of other objects, and the objects are classified (104). Here, an object close to the luminance ratio of the control probe object is recognized as a probe object, and a different object is recognized as another object. Then, the fluorescence luminance is calculated for the recognized area of the probe object (105). The procedure of the image information processing method in FIG. 1 will be described in detail later.

  FIG. 2 is a block diagram showing the configuration of the information processing apparatus applied to the image information processing apparatus according to the present invention. The image information processing apparatus is mounted on an apparatus including an external storage device 201, a central processing unit (CPU) 202, a memory 203, and an input / output device 204. The external storage device 201 holds a luminance level as a result of a program for realizing the present invention and a hybridization reaction. A central processing unit (CPU) 202 executes a program for realizing the present invention and controls all devices. The memory 203 temporarily stores programs used by the central processing unit (CPU) 202 and data such as subroutines, pixel defect data, and captured image data. The input / output device 204 interacts with the user. In many cases, a trigger for executing a program for realizing the biological species determination method of the present invention is issued by the user via this input / output device. The user's result confirmation and program parameter control are performed via this input / output device.

An image information processing apparatus according to the present invention is an image information processing apparatus that detects fluorescence intensity of probes on a probe array based on a hybridization reaction result in an image obtained by hybridization reaction of the probe array, separately from fluorescence intensity other than the probe. There,
Means for calculating a luminance ratio of red, green and blue pixels in the image subjected to the hybridization reaction, and means for discriminating between the luminance luminance of the probe and the fluorescent luminance other than the probe from the luminance ratio. Further, the image processing apparatus may further include means for recognizing the object, or may further include means for recognizing a pixel region having a luminance equal to or higher than a set threshold in an image subjected to a hybridization reaction. .

  FIG. 3 is a diagram showing a configuration of a fluorescence image pickup apparatus (probe array image pickup apparatus) according to the present invention. The fluorescence image pickup apparatus includes the information processing apparatus 301, the image pickup apparatus 303, the DNA microarray excitation light source 306, the DNA microarray support base 308 that supports the DNA microarray 307, and the dark box 302 that blocks external light. The The imaging device 303 includes a CMOS sensor 304 and a fluorescent filter 305. An RGB color filter is attached to the CMOS sensor 304. The microarray 307 that has undergone the hybridization reaction is excited by the excitation light source 306, and the emitted fluorescence is imaged by the imaging device 303 through the fluorescence filter 305. When the Cy3 fluorescent dye is used, the excitation light source 306 uses a laser with a wavelength of 532 nm. The imaging device 303 is operated from the input / output device of the information processing device 301 connected by a USB cable.

An image pickup apparatus according to the present invention is an apparatus for picking up an image obtained by a hybridization reaction of a probe array, and includes a probe array holding means, a means for irradiating the probe array with excitation light,
An area sensor for detecting fluorescence intensity on the probe array and at least the image information processing apparatus are provided.

  FIG. 4 shows the state of hybridization on the DNA microarray. In most cases, DNA has a double helix structure, and the bond between the two strands is realized by hydrogen bonding between bases. On the other hand, RNA often exists in a single strand. There are four types of bases in the case of DNA, ATGC, and in the case of RNA, AUGC. The base pairs capable of hydrogen bonding are A-T (U) and G-C pairs. In general, a hybridization reaction refers to a state in which single-stranded nucleic acid molecules are partially bound to each other through a partial base sequence in the nucleic acid molecule. In the reaction assumed in the present invention, the nucleic acid molecule (probe) attached to the upper substrate in FIG. 4 is shorter than the nucleic acid molecule in the lower sample. Therefore, when the nucleic acid molecule present in the sample contains the base sequence of the probe, this hybridization reaction is successful, and the target nucleic acid molecule in the sample is trapped on the DNA microarray.

Example 1
In this example, an experimental procedure of a hybridization reaction experiment using a DNA microarray will be described in detail. FIG. 5 shows the overall flow of the operation procedure using the DNA microarray. The “sample” 501 is a liquid or individual that should contain the nucleic acid of interest. For example, when the present invention is applied to identify the causative bacteria of infectious diseases, such as blood derived from animals such as humans and livestock, body fluids such as sputum, gastric juice, vaginal secretions, oral mucus, urine and feces Everything that seems to contain bacteria, such as effluent, becomes a sample. In addition, a medium that may cause contamination by bacteria, such as food poisoning, food subject to contamination, drinking water, and water in the environment such as hot spring water, may be used as a sample. In addition, animals and plants such as quarantine at the time of import / export are also subject to this.

Next, 501 samples are amplified using the 502 “biochemical amplification” method. For example, when the present invention is applied to identify the causative bacteria of an infectious disease, a target nucleic acid is amplified by a PCR method using a PCR reaction primer designed for 16s rRNA detection, or a PCR amplified product is used as the original. Further, it is prepared by conducting a PCR reaction or the like. Moreover, you may prepare by amplification methods, such as LAMP method other than PCR. Thereafter, the amplified sample or the sample 501 itself is labeled by various labeling methods for visualization. As the labeling substance, fluorescent substances such as Cy3, Cy5, and Rodamin are usually used. Further, in the experimental procedure of 502 biochemical amplification, a labeled molecule may be mixed.
In addition to the method of labeling the target nucleic acid molecule with a fluorescent substance, a method of labeling the fluorescent dye on the probe side, such as FRET (fluorescence resonance energy transfer) method or ETPH (excimer formation 2-probe nucleic acid hybridization) method, should be used. You can also. In addition, a fluorescent labeling method using an intercalator such as EtBr (ethidium bromide) inserted into a hybrid of a probe and a target nucleic acid molecule can also be used. In this example, a case where a labeling method for adding a label molecule to a nucleic acid is used will be described. The nucleic acid to which the labeled molecule has been added undergoes a hybridization reaction (505) with 504 DNA microarrays. This situation is as shown in FIG.
For example, when the present invention is applied to identify a causative bacterium of an infectious disease, the DNA microarray 504 is obtained by immobilizing a probe (artificial nucleic acid) having a base sequence specific to the causative bacterium on a substrate. The probe design of each bacterium is, for example, highly specific to the bacterium than the genome part coding for 16s rRNA, and is a hybridization that is sufficient and does not vary in consideration of Tm and the like in each probe base sequence. Perform so that sensitivity can be expected. The carrier (substrate) for fixing the probe of the DNA microarray 504 may be a flat substrate such as a glass substrate, a plastic substrate, or a silicon wafer. Further, even if a three-dimensional structure having irregularities, a spherical shape such as a bead, a rod shape, a string shape, a thread shape, or the like is used, the embodiment and the effect of the present invention are not affected.

  Usually, the surface of the substrate is treated so that the probe DNA can be immobilized. In particular, a product having a functional group introduced so that a chemical reaction is possible on the surface is a preferable form in terms of reproducibility because the probe is stably bound in the course of the hybridization reaction. Examples of the immobilization method used in the present invention include an example using a combination of a maleimide group and a thiol (-SH) group. That is, a thiol (-SH) group is bonded to the end of the nucleic acid probe, and the solid phase surface is treated so as to have a maleimide group, so that the thiol group of the nucleic acid probe supplied to the solid surface and the solid phase The maleimide group on the surface reacts to immobilize the nucleic acid probe. As a method for introducing the maleimide group, first, an aminosilane coupling agent is reacted with a glass substrate, and then the maleimide group is reacted by reacting the amino group with an EMCS reagent (N- (6-Maleimidocaproyloxy) succinimide: Dojin). Introduce. Introduction of SH groups into DNA can be performed by using 5′-Thiol-Modifier C6 (Glen Research) on an automatic DNA synthesizer. Examples of combinations of functional groups used for immobilization include a combination of an epoxy group (on a solid phase) and an amino group (end of a nucleic acid probe) in addition to the above-described combination of a thiol group and a maleimide group. Further, surface treatment with various silane coupling agents is also effective, and oligonucleotides into which functional groups capable of reacting with functional groups introduced with the silane coupling agents are used. Furthermore, a method of coating a resin having a functional group can also be used.

  After performing the hybridization reaction, the surface of the 504 DNA microarray is washed, the nucleic acid not bound to the probe is peeled off, and then (usually) dried, and the fluorescence of the DNA microarray reflecting the hybridization reaction of 505 Measure the amount. This amount of fluorescence is based on the fluorescent label added to the target nucleic acid molecule, and indicates that there is a target nucleic acid molecule trapped by hybridization with the probe on the DNA microarray by the hybridization reaction. Further, even with a labeling method other than adding a fluorescent label to the target nucleic acid molecule, the amount of fluorescence by the fluorescent label in each labeling method is measured. In order to measure the amount of fluorescence, the substrate of the DNA microarray is irradiated with excitation light, and a fluorescence image is picked up by a sensor (506). The probe fluorescence intensity of the obtained DNA microarray fluorescence image (507) is calculated. Here, what is desired to be obtained as a fluorescence image of a DNA microarray (probe array) obtained by a hybridization reaction is a fluorescence image containing only a fluorescence signal indicating the binding (hybrid formation) between the probe on the DNA microarray and the target nucleic acid molecule. In addition, it is desirable to remove a fluorescent signal derived from a foreign substance or a pixel defect that is not a signal indicating hybridization.

Next, the principle of a DNA microarray for identifying infectious bacteria will be described with reference to FIG. It is assumed that the DNA microarray shown in FIG. 6 is made for the purpose of specifying Staphylococcus aureus, for example.
The left column is a treatment sequence derived from a wild strain of S. aureus, and the right column is a treatment sequence derived from a wild strain of Escherichia coli. For example, it can be considered that the flow on the left is a flow for processing blood of a patient infected with Staphylococcus aureus, and the flow on the right is a flow for processing blood of a patient infected with Escherichia coli. Both basically perform the same processing. That is, first, DNA is extracted from, for example, blood or sputum of a bacterially infected patient. In this case, generally, human DNA derived from somatic cells of a patient may be included. When the extracted DNA is small, amplification is performed by a method such as PCR. In this case, a fluorescent substance or a substance capable of binding the fluorescent substance is generally mixed as a label. When amplification is not performed, using the extracted DNA, a fluorescent substance or a substance capable of binding the fluorescent substance is mixed as a label while forming a complementary strand, or the fluorescent substance or fluorescent substance is directly added to the extracted DNA as it is. A bindable substance is added as a label. Usually, when PCR amplification is performed, for the purpose of identifying the bacteria of an infectious disease, it is common to amplify a part of a base sequence constituting ribosomal RNA so-called 16s rRNA. In this case, the PCR primer for S. aureus on the left and the PCR primer for E. coli on the right are almost the same. More specifically, multiplex PCR is performed using a primer set that can amplify the 16s rRNA coding portion of any fungus.

  If a DNA microarray designed for the purpose of determining S. aureus works correctly, the left hybrid solution will react positively with the spot and the right hybrid solution will react negatively with the spot.

  In exactly the same way, if a DNA microarray designed for the purpose of determining the presence of E. coli works correctly, the left hybrid solution reacts negatively and the right hybrid solution produces spotless Responds positively. Of course, infectious bacteria may be determined using a DNA microarray in which several types of spots that react specifically with various bacteria are arranged at the same time.

  Hereinafter, the flow of the operation described with reference to FIG. 5 will be described by showing an example of a specific operation for identifying the causative bacteria of the infectious disease. The organism type determination method according to the present invention is not limited to the identification of the causative bacteria of the infectious disease described below, but is used for determination of human constitution such as MHC and analysis of DNA and RNA related to diseases such as cancer. May be.

<Preparation of probe DNA>
Nucleic acid sequences (In) (n is a number) shown in Table 1 were designed as probes for detecting Enterobacter cloacae. Specifically, the probe base sequence shown below was selected from the genome part coding 16s rRNA. These probe base sequence groups are designed to be highly specific to the bacteria and to be expected to have sufficient hybridization sensitivity with consideration of Tm and the like for each probe base sequence.

  In the probes shown in the table, a thiol group was introduced at the 5 ′ end of the nucleic acid after synthesis as a functional group for immobilizing the DNA microarray according to a conventional method. After introduction of the functional group, it was purified and lyophilized. The freeze-dried internal standard probe was stored in a -30 ° C freezer.

  Staphylococcus aureus (An), Staphylococcus epidermidis (Bn), Escherichia coli (Cn), Neisseria pneumoniae (Dn), Pseudomonas aeruginosa (En), Serratia bacteria (Fn), The probe sets shown in Table 2 below were designed in the same manner for Streptococcus pneumoniae (Gn), Haemophilus influenzae (Hn), and Enterococcus faecalis (Jn) (n is a number).

<Preparation of PCR Primer for sample amplification>
The nucleic acid sequences shown in Table 3 were designed as PCR primers for amplifying 16s rRNA nucleic acid (target nucleic acid) for detection of pathogenic bacteria. Specifically, a probe set that specifically amplifies the 16s rRNA-encoding genomic portion, that is, a primer with the same melting temperature as possible at both ends of the approximately 1500 base length 16s rRNA coding region is designed. did. Multiple primers were designed to simultaneously amplify mutant strains and multiple 16s rRNA coding regions on the genome.

The primers shown in the table are synthesized and purified by high performance liquid chromatography (HPLC), and 3 kinds of Forward Primer and 3 kinds of Reverse Primer are mixed so that each Primer concentration is 10 pmol / μl final concentration. Dissolved in TE buffer.

<Extraction of Enterobacter cloacae Genome DNA (model sample)>
(Preparation of microorganism culture & Genome DNA extraction)
First, the Enterobacter cloacae standard strain was cultured according to a conventional method. 1.0 ml (OD600 = 0.7) of this microorganism culture solution was collected in a 1.5 ml capacity microtube, and the cells were collected by centrifugation (8500 rpm, 5 min, 4 ° C.). After discarding the supernatant, 300 μl of Enzyme Buffer (50 mM Tris-HCl: pH 8.0, 25 mM EDTA) was added and resuspended using a mixer. The resuspended bacterial solution was recovered again by centrifugation (8500 rpm, 5 min, 4 ° C.). After discarding the upper fine particles, the following enzyme solution was added to the collected cells and resuspended using a mixer.
Lysozyme 50 μl (20 mg / ml in Enzyme Buffer)
N-Acetylmuramidase SG 50 μl (0.2 mg / ml in Enzyme Buffer)
Next, the bacterial solution resuspended by adding the enzyme solution was allowed to stand for 30 minutes in a 37 ° C. incubator to lyse the cell wall.
(Genome extraction)
Genome DNA extraction of the following microorganisms was performed using a nucleic acid purification kit (MagExtractor-Genome-: manufactured by TOYOBO). Specifically, first, 750 μl of the dissolving / adsorbing solution and 40 μl of magnetic beads were added to the pretreated microorganism suspension and vigorously stirred for 10 minutes using a tube mixer (step 1). Next, the microtube was set on a separation stand (Magical Trapper), left to stand for 30 seconds, the magnetic particles were collected on the wall of the tube, and the upper fine was discarded while being set on the stand (step 2). Next, 900 μl of the washing solution was added, and the mixture was stirred for about 5 seconds with a mixer and re-suspended (step 3). Next, a microtube was set on a separation stand (Magical Trapper), left still for 30 seconds, magnetic particles were collected on the wall surface of the tube, and the upper fine was discarded while being set on the stand (step 4). Steps 3 and 4 were repeated to perform the second washing (Step 5), and then 900 μl of 70% ethanol was added and stirred for about 5 seconds with a mixer to re-suspend (Step 6). Next, the microtube was set on a separation stand (Magical Trapper), left to stand for 30 seconds, the magnetic particles were collected on the wall of the tube, and the upper fine was discarded while being set on the stand (step 7). Steps 6 and 7 were repeated and the second washing with 70% ethanol (step 8) was performed. Then, 100 μl of pure water was added to the collected magnetic particles, and the mixture was stirred for 10 minutes with a tube mixer. Next, the microtube was set on a separation stand (Magical Trapper), allowed to stand for 30 seconds to collect the magnetic particles on the wall of the tube, and the supernatant was collected in a new tube while being set on the stand.
(Examination of recovered Genome DNA)
Genome DNA of recovered microorganisms (Enterobacter cloacae strain) was subjected to agarose electrophoresis and absorbance measurement at 260/280 nm according to standard methods, and the quality (contamination amount of low molecular nucleic acid, degree of degradation) and recovery amount were tested. . In this example, about 10 μg of Genome DNA was recovered, and no degradation of Genome DNA or contamination with rRNA was observed. The recovered Genome DNA was dissolved in TE buffer so as to have a final concentration of 50 ng / μl and used in the following examples.

<Production of DNA microarray>
[1] Cleaning of Glass Substrate A synthetic quartz glass substrate (size: 25 mm × 75 mm × 1 mm, manufactured by Iiyama Special Glass Co., Ltd.) was placed in a heat-resistant and alkali-resistant rack and immersed in a cleaning solution for ultrasonic cleaning adjusted to a predetermined concentration. After soaking in the cleaning solution overnight, ultrasonic cleaning was performed for 20 minutes. Subsequently, the substrate was taken out, rinsed lightly with pure water, and then ultrasonically cleaned in ultrapure water for 20 minutes. Next, the substrate was immersed in a 1N sodium hydroxide aqueous solution heated to 80 ° C. for 10 minutes. Pure water cleaning and ultrapure water cleaning were performed again to prepare a quartz glass substrate for a DNA chip.
[2] Surface treatment A silane coupling agent KBM-603 (manufactured by Shin-Etsu Silicone) was dissolved in pure water to a concentration of 1% and stirred at room temperature for 2 hours. Subsequently, the previously cleaned glass substrate was immersed in an aqueous silane coupling agent solution and allowed to stand at room temperature for 20 minutes. The glass substrate was pulled up and the surface was lightly washed with pure water, and then nitrogen gas was blown onto both sides of the substrate to dry it. Next, the dried substrate was baked in an oven heated to 120 ° C. for 1 hour to complete the coupling agent treatment, and amino groups were introduced onto the substrate surface. Next, N- (6-Maleimidocaproyloxy) succinimido (hereinafter abbreviated as EMCS) manufactured by Dojindo Laboratories Ltd. was added to a 1: 1 mixed solvent of dimethyl sulfoxide and ethanol. An EMCS solution dissolved to 0.3 mg / ml was prepared. The glass substrate after baking was allowed to cool and immersed in the prepared EMCS solution for 2 hours at room temperature. By this treatment, the amino group introduced to the surface by the silane coupling agent and the succinimide group of EMCS reacted, and the maleimide group was introduced to the glass substrate surface. The glass substrate pulled up from the EMCS solution was washed with the above-mentioned mixed solvent in which MCS was dissolved, further washed with ethanol, and then dried in a nitrogen gas atmosphere.
[3] Probe DNA
The microorganism detection probe prepared in <Preparation of probe DNA> was dissolved in pure water and dispensed to a final concentration (at the time of ink dissolution) of 10 μM, and then freeze-dried to remove moisture.
[4] DNA ejection by inkjet printer and binding to substrate An aqueous solution containing glycerin 7.5 wt%, thiodiglycol 7.5 wt%, urea 7.5 wt%, and acetylenol EH (manufactured by Kawaken Fine Chemical Co., Ltd.) 1.0 wt% was prepared. Subsequently, the six kinds of probes prepared previously (Table 1) were dissolved in the above mixed solvent so as to have a prescribed concentration. The obtained DNA solution was filled in an ink tank for an ink jet printer (trade name: BJF-850 manufactured by Canon Inc.) and mounted on a print head.
The ink jet printer used here has been modified so that printing on a flat plate is possible. This ink jet printer can spot a DNA solution of about 5 picoliters at a pitch of about 120 micrometers by inputting a print pattern according to a predetermined file creation method.

Subsequently, using this modified inkjet printer, a printing operation was performed on one glass substrate to produce an array. After confirming that printing was performed reliably, the sample was left in a humidified chamber for 30 minutes to react the maleimide group on the surface of the glass substrate with the thiol group at the end of the nucleic acid probe.
[5] Cleaning
After the reaction for 30 minutes, the DNA solution remaining on the surface was washed away with 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl to obtain a DNA microarray in which single-stranded DNA was immobilized on the surface of the glass substrate.

<Amplification and labeling of specimen (PCR amplification & incorporation of fluorescent label)>
Amplification of microbial DNA as a specimen and labeling reaction are shown below.
Premix PCR reagent (TAKARA ExTaq) 25μl
Template Genome DNA 2μl (100ng)
Forward Primer mix 2μl (20 pmol/tube each)
Reverse Primer mix 2μl (20 pmol/tube each)
Cy-3 dUTP (1mM) 2μl (2nmol / tube)
H ₂ 0 17μl
Total 50μl
The reaction solution having the above composition was subjected to an amplification reaction using a commercially available thermal cycler according to the protocol of FIG. After completion of the reaction, Primer was removed using a purification column (QIAGEN QIAquick PCR Purification Kit), and then the amplification product was quantified to obtain a labeled sample.

<Hybridization>
Detection reaction was performed using the DNA microarray prepared in <Preparation of DNA microarray> and the labeled sample prepared in <Amplification and labeling of specimen (PCR amplification & incorporation of fluorescent label)>.
(Blocking of DNA microarray)
BSA (bovine serum albumin Fraction V: manufactured by Sigma) was dissolved in 100 mM NaCl / 10 mM Phosphate Buffer so as to be 1 wt%. The DNA microarray prepared in <Preparation of DNA microarray> was immersed in this solution at room temperature for 2 hours for blocking. After blocking, wash with 2xSSC solution (NaCl 300mM, Sodium Citrate (trisodium citrate dihydrate, C6H5Na3 · 2H2O) 30mM, pH 7.0) containing 0.1wt% SDS (sodium dodecyl sulfate) and rinse with pure water. The water was drained with a spin dryer.
(Hybridization)
The drained DNA microarray was set in a hybridization apparatus (Genomic Solutions Inc. Hybridization Station). And hybridization reaction was performed with the following hybridization solution and conditions.
Hybridization solution;
6 x SSPE / 10% Form amide / Target (2nd PCR Products total amount)
(6xSSPE: NaCl 900mM, NaH2PO4 ・ H2O 60mM, EDTA 6mM, pH 7.4)
Hybridization conditions;
65 ° C 3 min → 92 ° C 2 min → 45 ° C 3 hr → Wash 2 x SSC / 0.1% SDS at 25 ° C → Wash 2 x SSC at 20 ° C → (Rinse with H2O: Manual) → Spin dry
<Detection of microorganisms (fluorescence measurement)>
The DNA microarray after completion of the hybridization reaction was subjected to fluorescent image capturing using the apparatus of FIG. The resulting fluorescent image derived from Staphylococcus aureus is shown in FIG. Here, the presence / absence of Staphylococcus aureus is determined by quantifying the fluorescence luminance of the probe spot.

  In the same manner as described above for Staphylococcus aureus, the presence or absence of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Neisseria pneumoniae, Pseudomonas aeruginosa, Serratia, Streptococcus pneumoniae, Haemophilus influenzae, and Enterococcus faecalis judge. That is, the presence or absence of each bacterium is determined by performing a hybridization reaction using a DNA microarray to which the probe set shown as the probe set of these bacterium is fixed.

(Example 2)
Hereinafter, each step of the image information processing (FIG. 1) for distinguishing between the probe and the foreign matter / pixel defect based on the luminance ratio of the present invention will be described in detail.

  FIG. 8 is a diagram showing a foreign substance, a pixel defect, and a probe in a DNA microarray fluorescence image, which corresponds to a detailed explanation of 101 in FIG. Reference numeral 801 denotes an example of an RGB composite color image of a DNA microarray fluorescence image captured by an area sensor equipped with an RGB color filter. The fluorescence of the probe is due to the same Cy3 fluorescent dye as in Example 1. 802 is an enlarged version of the probe. In an RGB composite color image, the red and green components are almost equal and appear yellow. Reference numerals 803 and 804 are enlarged foreign objects. As for the brightness | luminance of the foreign material image of 803,804, the red component is strong as a whole. 805 has a luminance of only a blue component due to a pixel defect. Similarly, there are pixel defects that have a luminance of only the red component and those that do not emit luminance at all. As described above, since the luminance ratio of RGB other than the probe and the probe is different, the fluorescence luminance other than the probe and the probe is distinguished using this.

  FIG. 9 is a diagram for explaining a method of recognizing an object from the magnitude of luminance, and is a detailed explanation of 102 in FIG. A constant luminance threshold is set for the fluorescent image captured at 101 in FIG. As shown in the luminance cross-sectional view, a pixel having a luminance equal to or higher than a threshold at a boundary with a pixel having a luminance lower than the threshold is defined as an object edge. Then, an area surrounded by edges is defined as an object. Although the object recognition method based on the threshold is shown here, other methods such as a differential processing method may be used for edge detection.

  FIG. 10 is a diagram for explaining a method of interpolating B and R pixels with G pixel values. In an image captured by an area sensor equipped with an RGB color filter, luminance values of red, green, and blue are stored in RGB pixels. Here, in order to perform edge detection with a green luminance value, the red and blue pixels are respectively interpolated with the average value of the green luminance values surrounding the red and blue pixels. The blue luminance value B1 in FIG. 10 is an average value of G1, G2, G3, and G4 (Formula 1). Similarly, B2 is an average value of G5, G4, G6, and G7 (Formula 2). The red luminance value R1 is an average value of G4, G5, G10, and G6 (Formula 3). Similarly, R2 is an average value of G7, G6, G11, and G9 (Formula 4). In this way, the luminance values for edge detection can be obtained by interpolating the luminance values of red and blue with the luminance values of the surrounding green.

B1 = (G1 + G2 + G3 + G4) ÷ 4 (Equation 1)
B2 = (G5 + G4 + G6 + G7) ÷ 4 (Equation 2)
R1 = (G4 + G5 + G10 + G6) ÷ 4 (Equation 3)
R2 = (G7 + G6 + G11 + G9) ÷ 4 (Equation 4)
An image information processing apparatus using the above-described interpolation method can be used for processing a hybridized image of the probe array. An image information processing apparatus that interpolates pixels whose luminance is saturated in the hybridized image of the probe array includes means for calculating the luminance ratio of red, green, and blue pixels in the hybridized image, and the luminance from the luminance ratio. And at least means for calculating luminance of saturated pixels. Moreover, it can also be set as a probe array image pick-up device provided with this image information processing apparatus. An apparatus that captures an image obtained by performing a hybridization reaction of a probe array includes a probe array holding unit, a unit that irradiates the probe array with excitation light, an area sensor that detects fluorescence intensity on the probe array, and the image information processing apparatus. And at least.

  FIG. 11 is a diagram for explaining a method of calculating the object area. As described with reference to FIG. 9, a pixel having a luminance equal to or higher than a threshold value at a boundary with a pixel having a luminance lower than the threshold value is defined as an object edge, and a region surrounded by the edge is defined as an object region. At this time, even if there are pixels below the threshold in the area surrounded by the edges, they are included in the object area.

FIG. 12 is a diagram for explaining a method for calculating the luminance ratio of the object, and corresponds to explanation 103 in FIG. When the table in FIG. 12 is the pixel of the object area, the R / G ratio and the B / G ratio are calculated as follows as the luminance ratio of the object.
R / G ratio [%] = average value of R ÷ average value of G = 82.94 [%]
B / G ratio [%] = B average value ÷ G average value = 1.88 [%]
The luminance ratio calculation of the object is performed for all the pixels in the object area. The brightness ratio calculation of the object is performed for all objects in the image.

FIG. 13 is a diagram for explaining a method of classifying objects by comparing the RGB luminance ratio of the control object with the RGB luminance ratio of the object other than the control, and corresponds to the detailed description of 104 in FIG. In FIG. 13, the upper right object in the image is set as the control object. As shown in FIG. 12, by calculating the luminance ratio of all objects, comparing the luminance ratio of the control object and the luminance ratio of the objects other than the control, and determining whether they are within a certain range, Classify objects. When the probe object condition is within the control object R / G ratio [%] ± 5 [%] and the control object B / G ratio [%] ± 0.5 [%] It can be calculated as follows.
R / G ratio of control object [%] = 82.94 [%]
B / G ratio of control object [%] = 1.88 [%]
77.94 [%] ≤ R / G ratio of object [%] ≤ 87.94 [%] ・ ・ ・ ・ （Formula 5）
1.33 [%] ≤ Object B / G ratio [%] ≤ 2.38 [%] ... (Formula 6)
When the conditions of the expressions (5) and (6) are satisfied at the same time, it is determined as a probe object. When even one of the conditions of Expressions (5) and (6) is not satisfied, it is determined as an object other than the probe due to dust, dirt, pixel failure, and the like. Finally, the target probe luminance is obtained by calculating the average luminance of the green pixels in the probe object area (105 in FIG. 1).

  FIG. 14 is a flowchart showing the procedure of luminance value correction processing for luminance saturated pixels. The luminance correction processing uses the fact that the RGB luminance ratio becomes a substantially constant ratio, and corrects the luminance value of the pixel (G) whose luminance is saturated using the values of other adjacent pixels (R, B). It is processing. The same processing as steps 101, 102, and 103 of the image information processing method in FIG. The process 103 in FIG. 1 corresponds to 1401 in FIG. The brightness ratio of the control object obtained here is used for brightness value correction. Next, a luminance saturated pixel in the object area is searched (1402). A luminance saturated pixel is a pixel whose luminance could not be measured any more due to the upper limit of the dynamic range of the measured luminance. Finally, the luminance value of the luminance saturated pixel is corrected using the luminance ratio of the control object obtained in 1401.

FIG. 15 is a diagram for explaining a luminance value correction method for luminance saturated pixels. When the green pixel G1 is saturated and the adjacent red pixels R1 and R2 are not saturated, the corrected luminance value of G1 is obtained by dividing the average value of R1 and R2 by the R / G ratio of the control object (Formula 7). When the green pixel G2 is saturated and the adjacent red pixel R3 is also saturated, the corrected luminance value of G2 is obtained by dividing the average value of the blue pixels B1 and B2 by the B / G ratio of the control object. (Equation 8). The corrected luminance value of the red pixel R3 is obtained by dividing the average value of the blue pixels B1, B2, B3, and B4 by the B / R ratio of the control object. When correcting the luminance value of green, the luminance value of the blue pixel is smaller than the luminance value of the green or red pixel, so if the luminance value of the red pixel is not saturated, the luminance value of the red pixel It is desirable to correct using
G1 = (R1 + R2) ÷ 2 ÷ R / G ratio of control object (Equation 7)
G2 = (B1 + B2) ÷ 2 ÷ B / G ratio of control object (Equation 8)
R3 = (B1 + B2 + B3 + B4) ÷ 4 ÷ B / R ratio of control object (Equation 9)
FIG. 16 is a diagram for explaining a method of calculating a luminance value correction value of a luminance saturated pixel. The luminance value (G: 3967) of the luminance saturated pixel before luminance correction is corrected. A calculation example of the luminance correction value of G1 using the luminance values of R1 and R2 is shown below (Formula 10).
R / G ratio of control object [%] = 82.94 [%]
G1 = (R1 + R2) ÷ 2 ÷ R / G ratio of control object
= (3209 + 3519) ÷ 2 ÷ 0.8294
= 4056 ・ ・ (Formula 10)

It is the figure which showed the flow of the image information processing which distinguishes the probe by a luminance ratio of this invention, and a foreign material and pixel defect. It is a block diagram which shows the structure of the information processing apparatus for implement | achieving this invention. It is the figure which showed the structure of the probe array image imaging device of the fluorescence image for implement | achieving this invention. It is the figure which showed the mode of hybridization on a DNA microarray. It is a figure for demonstrating the whole experimental procedure of the hybridization reaction experiment using a DNA microarray. It is a figure for demonstrating the principle of the DNA microarray which identifies the microbe of an infectious disease. It is the figure which showed the DNA microarray fluorescence image derived from Staphylococcus aureus. It is the figure which showed the foreign material, pixel defect, and probe of a DNA microarray fluorescence image. It is a figure for demonstrating the method of recognizing an object from the magnitude | size of a brightness | luminance. It is a figure for demonstrating the method of interpolating the pixel of B and R by the pixel value of G. It is a figure for demonstrating the calculation method of an object area | region. It is a figure for demonstrating the calculation method of the luminance ratio of an object. It is a figure for demonstrating the method of classifying an object by comparing the RGB luminance ratio of a control probe with the RGB luminance ratio of an object. It is the figure which showed the flow of the luminance value correction process of a luminance saturation pixel. It is a figure for demonstrating the luminance value correction method of a luminance saturation pixel. It is a figure for demonstrating the calculation method of the luminance value correction value of a luminance saturation pixel. It is a figure which shows the protocol of PCR amplification reaction.

Explanation of symbols

101 DNA microarray fluorescent image imaging step 102 Object recognition step from luminance magnitude 103 Object RGB luminance ratio calculation step 104 Object classification step 105 Probe object luminance calculation step 201 External storage device 202 Central processing unit (CPU)
203 Memory 204 Input / Output Device 301 Information Processing Device 302 Dark Box 303 Imaging Device 304 CMOS Sensor 305 Fluorescent Filter 306 Excitation Light Source 307 DNA Microarray 308 DNA Microarray Support Base 501 Sample 502 Biochemical Amplification 503 Label Contamination 504 DNA Microarray 505 Hybridization Reaction 506 Imaging 507 DNA microarray fluorescence image 801 DNA microarray fluorescence image (RGB composite color image)
802 Probe fluorescence image 803 Foreign object image 804 Foreign object image 805 Pixel defective image 1401 Object RGB luminance ratio calculation step 1402 Luminance saturated pixel search step 1403 Luminance saturated pixel luminance value correction step

Claims (9)

An image information processing apparatus for detecting the fluorescence intensity of probes on a probe array based on the hybridization reaction result separately from the fluorescence intensity other than the probe in an image obtained by performing a hybridization reaction of a probe array,
Means for calculating a luminance ratio of red, green and blue pixels in the image;
Means for distinguishing the fluorescence brightness of the probe and the fluorescence brightness other than the probe from the brightness ratio;
An image information processing apparatus comprising:   The image information processing apparatus according to claim 1, further comprising means for recognizing a pixel region having a luminance equal to or higher than a set threshold in the image subjected to the hybridization reaction.   The image information processing apparatus according to claim 1, wherein the probe array is a DNA microarray. An apparatus for taking an image of a probe array hybridization reaction,
Probe array holding means;
Means for irradiating the probe array with excitation light;
An area sensor for detecting fluorescence intensity on the probe array;
An image information processing apparatus according to any one of claims 1 to 3,
A probe array imaging apparatus comprising:   The image capturing apparatus according to claim 4, wherein the area sensor is a CMOS sensor. An image information processing apparatus that interpolates pixels with saturated brightness in a hybridized image of a probe array,
Means for calculating a luminance ratio of red, green and blue pixels in the image;
Means for calculating the luminance of the pixel whose luminance is saturated from the luminance ratio;
An image information processing apparatus comprising:   The image pickup apparatus according to claim 4, wherein the probe array is a DNA microarray. An apparatus for taking an image of a probe array hybridization reaction,
Probe array holding means;
Means for irradiating the probe array with excitation light;
An area sensor for detecting fluorescence intensity on the probe array;
An image information processing apparatus according to claim 6 or 7,
A probe array imaging apparatus comprising:   The image capturing apparatus according to claim 8, wherein the area sensor is a CMOS sensor.